Tyrosine phosphorylation, long studied in eukaryotes, is beginning to be recognized as playing a key regulatory role in bacterial physiology. This process is catalyzed by a novel class of protein tyrosine kinases, the BY- kinases, which are conserved in both Gram-negative and Gram-positive bacteria, but are distinct from their eukaryotic counterparts, with unique structural and regulatory features. BY-kinase mediated signal transduction plays a central role in the synthesis of polysaccharides responsible for capsule and biofilm formation, the former acting as antiphagocytic virulence factors for many pathogenic species, and the latter protecting the cell from a variety of environmental stresses. The regulatory module for BY-kinases is a cluster of tyrosine residues (YC) in the C-terminal tail of its cytosolic catalytic domain. The YC is autophosphorylated in an intermolecular fashion and dephosphorylated by low molecular weight protein tyrosine phosphatases (LMW-PTP). Unlike eukaryotic protein kinases which act as active/inactive or on-off switches dependent on the quantitative phosphorylation states of one or more regulatory residues, in BY-kinases both phosphorylated and dephosphorylated species are required for optimal polysaccharide synthesis. Therefore it appears that the dynamic equilibrium between phosphorylated and dephosphorylated forms, resulting from a balance between the kinase and phosphatase activities, is key to proper cellular function. Thus, determining the nature of the interactions between BY-kinases and LMW-PTPs is of fundamental importance in fully understanding the physiochemical aspects of tyrosine phosphorylation mediated signaling in bacteria. Although the structures of several BY-kinase catalytic domains and bacterial LMW-PTPs are available, these do not provide any insight into the nature of their mutual interactions or the physiological consequences of disrupting them. Using the catalytic domain of Wzc, an E. coli (K12) BY-kinase and Wzb, the corresponding LMW-PTP, as a model kinase-phosphatase system, and utilizing state-of-the-art solution NMR methods combined with site-directed mutagenesis, we will ascertain the key residues that define the kinase- phosphatase interface. The relevance of these NMR-determined interactions in the context of the physiology of the intact host organism will be ascertained by assaying cells expressing mutant proteins for polysaccharide synthesis and survival under desiccation. We expect that our present studies, limited to the E. coli Wzc/Wzb system, will facilitate the development of a more elaborate research project involving other bacterial BY-kinase/LMW-PTP pairs including those from pathogenic species, and the construction of a comprehensive mechanistic model of BY-kinase mediated tyrosine phosphorylation in bacteria.